Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: Electrical properties of DGGCs

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: shRNA, Membrane

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: a The averaged current – voltage (I–V) relationship of the whole-cell currents from DGGCs infected with Ad-Sc or Ad-TASK-3 shRNAs or both Ad-TASK-3 shRNA and TWIK-1 shRNA as well as from naive DGGCs was measured in standard artificial cerebrospinal fluid in the presence of Cs + /TEA/4-AP (1 mM/5 mM/5 mM). Whole-cell currents were elicited by 1-s-duration ramp pulses descending from 50 mV to −150 mV from a holding potential of −70 mV. b A summary bar graph for a . The mean values of the current density in naive DGGCs ( n = 14 cells, N = 4 mice) or DGGCs expressing Sc shRNA ( n = 12 cells, N = 3 mice), TASK-3 shRNA ( n = 15 cells, N = 3 mice), or both TASK-3 and TWIK-1 shRNAs ( n = 15 cells, N = 3 mice) measured in the presence of Cs + /TEA/4-AP (1 mM/5 mM/5 mM) are shown. The current density values are depicted at +50 mV. c The TASK-3 shRNA- or both TASK-3 shRNA- and TWIK-1 shRNA-sensitive currents were determined by subtracting each of the shRNA averaged currents from the Sc shRNA averaged currents a . ** P < 0.01

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: Infection, shRNA, Expressing

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: a Representative traces of the membrane potential to stepwise current injections recorded from naive DGGCs ( n = 27, N = 3) or DGGCs infected with Ad-Sc shRNA ( n = 21, N = 3), Ad-TWIK-1 shRNA ( n = 30, N = 3), Ad-TASK-3 shRNA ( n = 22, N = 3), or both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNA ( n = 32, N = 3). The resting membrane potentials of the cells was maintained at −70 mV by constant current injections, and the depolarizing current was then injected stepwise in 5-pA increments. b The number of spikes indicated that the neuron infected with Ad-TWIK-1 shRNA and Ad-TASK-3 shRNA were more excitable compared to control mice. The recordings were performed in artificial cerebrospinal fluid containing 50 µM D-AP5, 10 µM CNQX, 10 µM bicuculline, 10 µM CGP 55845, 2 mM TEA, and 0.5 mM NiCl 2 , with a pipette solution containing 5 mM QX314. c Averaged values of rheobase currents in naive cells ( n = 27 cells, N = 3 mice) and cells expressing Ad-Sc shRNA ( n = 21 cells, N = 3 mice), Ad-TWIK-1 shRNA ( n = 30 cells, N = 3 mice), Ad-TASK-3 shRNA ( n = 12), or both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNA ( n = 32 cells, N = 3 mice). All values are means ± SEM. * P < 0.05, *** P < 0.001

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: Membrane, Infection, shRNA, Injection, Control, Transferring, Expressing

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: a Bath application of NT-mediated membrane depolarization of granule cells. A representative response of the membrane potential to stepwise current injections was recorded from naive DGGCs ( n = 15, N = 3) or DGGCs infected with Ad-Sc shRNA ( n = 16, N = 3), Ad-TWIK-1 shRNA ( n = 17, N = 3), Ad-TASK-3 shRNA ( n = 15, N = 3), or both Ad-TWIK-shRNA and Ad-TASK-3 shRNA ( n = 14, N = 3). The resting membrane potential of these cells was maintained at −70 mV by constant current injections, and depolarizing currents were then injected stepwise at 5-pA increments until the membrane potential reached the firing threshold. b , c Analyzed bar charts of spike numbers at 105 pA for a . b Resting membrane potential values of the whole-cell currents in naive dentate gyrus granule cells ( n = 15 cells, N = 3 mice) and cells expressing Sc shRNA ( n = 16 cells, N = 3 mice), TWIK-1 shRNA ( n = 17 cells, N = 3 mice), TASK-3 shRNA ( n = 15 cells, N = 3 mice), or TWIK-1/TASK-3 shRNAs ( n = 14 cells, N = 3 mice). c The extent of changes in the number of spikes following NT application in naive and Ad-Sc shRNA-infected DGGCs was larger than those in DGGCs transfected with Ad-TASK-1 shRNA alone, Ad-TWIK-1 shRNA alone, or both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNAs. The data for Ad-TASK-3 shRNA alone, Ad-TWIK-1 shRNA alone, and both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNAs showed no change in the numbers of spikes. The recordings were obtained in artificial cerebrospinal fluid containing 50 µM D-AP5, 10 µM CNQX, 10 µM bicuculline, 10 µM CGP55845, 2 mM TEA, and 0.5 mM NiCl 2 , with a pipette solution containing 5 mM QX314. All the data are presented as the means ± SEM. *** P < 0.001 was considered statistically significant

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: Membrane, Infection, shRNA, Injection, Expressing, Transfection, Transferring

Journal: PLoS Biology

Article Title: Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis

doi: 10.1371/journal.pbio.3001599

Figure Lengend Snippet: (A–D) Live spermatocytes were incubated with purified PlyA2-mCherry (10 μg/ml) for 30 min followed by washes with M3 insect media (2×) and imaged using spinning disk confocal microscopy. (A and B) w 1118 spermatocytes, (C and D) cpes spermatocytes. (E–P) Live spermatocytes expressing endogenous EYFP tagged Rab proteins were incubated with PlyA2-mCherry for 1 h followed by washes with M3 insect media (2×) and imaged using spinning disk confocal microscopy. (E–G) EYFP-Rab4 spermatocytes, (H–J) EYFP-Rab6 spermatocytes, (K–M) EYFP-Rab7 spermatocytes, (N–P) EYFP-Rab11 spermatocytes. Arrowheads indicate colocalizing structures. (Q) Colocalization coefficient (Mander’s coefficient) measurements showing fraction of EYFP Rab proteins colocalize with internalized PlyA2-mCherry. Mander’s coefficient was determined using ImageJ plugin JACoP. Each dot in the graph represents a 2D image consisting of 8–16 spermatocytes, Rab4 ( n = 42), Rab6 ( n = 47), Rab7 ( n = 36), and Rab11 ( n = 81). Statistical significance was calculated using mean, SD, and N in Prism 9. The ordinary 1-way ANOVA multiple comparison was used to calculate P values where **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, and ns P >0.05. The data underlying the graph shown in this figure can be found in . CPE, ceramide phosphoethanolamine; SD, standard deviation.

Article Snippet: Briefly, the coding sequence of PlyA2 (accession number AB777517) fused to mCherry at the C-terminus was synthesized by GenScript.

Techniques: Incubation, Purification, Confocal Microscopy, Expressing, Comparison, Standard Deviation

Journal: PLoS Biology

Article Title: Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis

doi: 10.1371/journal.pbio.3001599

Figure Lengend Snippet: (A–F) The EYFP-Rab7 expressing spermatocytes are treated with PlyA2-mCherry and snapshots of spermatocytes undergoing meiosis I cytokinesis are shown. (A–C) Horizontal view of 1 z slice is shown. (D–F) Cross-sectional view of a spermatocyte undergoing cytokinesis, maximum intensity projection image was shown. (G) Quantification of endosomal volume localized to cleavage furrow in EYFP-Rab7 expressing and PlyA2-mCherry treated spermatocytes. Using surfaces tool in Imaris software, we have calculated the total endosomal volume and fraction of which localized to cleavage furrow in 3D images ( n = 6 spermatocytes from 3 independent experiments). (H–J) Localization of EYFP-Rab7 in intact cysts where spermatocytes are encapsulated by cyst cells. Horizontal view of a single z slice is shown. (K–P) EYFP-Rab11 expressing spermatocytes were treated with PlyA2-mCherry and meiosis I cytokinesis was imaged. (K–M) Horizontal view of single z slice is shown. (N–P) Cross-sectional view, maximum intensity projection image is shown. White arrows indicate colocalization and white arrowheads indicate lack of colocalization. (Q) Quantification of a fraction of endosomal volume localized to cleavage furrow in EYP-Rab11 expressing, PlyA2-mCherry treated spermatocytes. Endosomal volume in 3D images were calculated using surfaces tool in Imaris software ( n = 9 spermatocytes from 3 independent experiments). The data underlying the graphs shown in this figure can be found in . CPE, ceramide phosphoethanolamine.

Article Snippet: Briefly, the coding sequence of PlyA2 (accession number AB777517) fused to mCherry at the C-terminus was synthesized by GenScript.

Techniques: Expressing, Software

Journal: PLoS Biology

Article Title: Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis

doi: 10.1371/journal.pbio.3001599

Figure Lengend Snippet: (A) Combined transmitted and fluorescence image of dissected Drosophila cyst; dividing cells chosen for FIB-SEM imaging are boxed. Green, eYFP-Rab7; red, PlyA2-mCherry. Scale bar: 20 μm. (B) 2D section from FIB-SEM reconstruction correlated with “z slice” from confocal. Colors as in A, saturated for ease of visualization. Scale bar: 5 μm. (C) Area boxed in B; FIB-SEM image (top), Rab7 (middle), PlyA2 (bottom). Arrowheads indicate electron-dense endosomes. Scale bar: 2 μm. (D) Volumetric imaging of dividing spermatocytes, showing orthogonal imaging planes, and expected cross sections for LM and FIB-SEM. (E) Fluorescence “z slice” capturing a cleavage furrow in-plane. Scale bar: 4 μm. (F) FIB-SEM image capturing same furrow as in E cross-section, approximated by red lines. Scale bar: 2 μm. (G–I) Correlated fluorescence image, overlay, and rotated FIB-SEM section, respectively, of furrow in-plane. (J) Area boxed in I showing endosomes docked at the furrow (arrow). Scale bar: 1 μm. (J and K) Volume rendering of FIB-SEM 3D reconstruction, with furrow segmented and false colored red, undocked endosomes green, docked endosomes blue. (L) Close-up of K, with furrow segmented translucent and endosomes unsegmented. (M) Matching FIB-SEM section in the imaging plane. Arrows in K, L, M show same endosomes docked at furrow. Scale bar: 2 μm. (N) Independent CLEM/FIB-SEM experiment revealing at higher pixel sampling another example of docked vesicle. Scale bar: 1 μm. CPE, ceramide phosphoethanolamine; LM, light microscopy.

Article Snippet: Briefly, the coding sequence of PlyA2 (accession number AB777517) fused to mCherry at the C-terminus was synthesized by GenScript.

Techniques: Fluorescence, Imaging, Sampling, Light Microscopy

Journal: PLoS Genetics

Article Title: Balanced Codon Usage Optimizes Eukaryotic Translational Efficiency

doi: 10.1371/journal.pgen.1002603

Figure Lengend Snippet: (A) Experimental design for examining the impact of mCherry expression on the expression of the reporter vYFP. An mCherry gene is constitutively expressed from a 2-micron plasmid in S. cerevisiae , whereas vYFP is constitutively expressed from Chromosome XII. Four different synonymous versions of mCherry are compared. (B) The codon adaptation indices ( CAI s) of the four synonymous mCherry sequences (circled numbers), in comparison to CAI s of all S. cerevisiae genes. (C) Values of distance to native codon usage of yeast ( D ncu ) for the four mCherry sequences, in comparison to that of all S. cerevisiae genes. (D) Relationship between vYFP expression and the CAI or D ncu of mCherry , when the mCherry expression is controlled for. A finer control of mCherry expression is presented in , where cells of the low, intermediate, and high mCherry expressions defined here are each subdivided into 5 bins. Error bars, which are barely seen, show one standard error. (E) vYFP expressions in the four strains after the removal of the plasmids that carry mCherry . Error bars show one standard error. (F) vYFP mRNA levels of the four strains relative to that of the wild-type strain, which does not carry mCherry . The mean expressions from three biological replications and the standard errors are presented.

Article Snippet: The different versions of mCherry DNA sequences were synthesized by Blue Heron Biotechnology.

Techniques: Expressing, Plasmid Preparation, Comparison, Control

Journal: Technology in Cancer Research & Treatment

Article Title: In Vivo Visualized Tracking of Tumor-Derived Extracellular Vesicles Using CRISPR-Cas9 System

doi: 10.1177/15330338221085370

Figure Lengend Snippet: The design and characterization of EV tracking system. (A) Schematic of EV tracking system. This system consisted of donor A549 cells that expressed sgRNAs and Cas9 proteins and recipient cells that contained STOP-FP elements. (B) Schematic of the recuperation of FP expression in reporter cells. (C) Schematic of engineered EVs. FP Nb and its affinitive Nb were fused with exosomal membrane protein CD63 and Cas9 proteins, respectively. Cas9 proteins were captured by EVs through the bond of FP-FP Nb, selectively being sorted into EVs. (D) qRT-PCR analysis of sgRNA pair levels in EVs derived from wild-type A549 or donor A549 cells (n = 3 in each group). (E) Western blot analysis of Cas9 protein levels in EVs derived from wild-type A549 cells or donor A549 cells (n = 3 in each group). (F) RNA immunoprecipitation (IP) assay of exosomal Cas9 and sgRNAs derived from wild-type A549 cells or donor A549 cells. Cas9 proteins were tagged with Flag. Lysates of EVs were blotted using Flag antibody (input) or immunoprecipitated with Flag and blotted using Flag antibody (IP). IP with anti-IgG served as control. (G) sgRNAs analysis of immunoprecipitants following IP assay in (F). Abbreviations: EV: extracellulaer vesicle; FP: fluorescent protein; Nb: nanobody; sgRNAs: single-guide RNAs.

Article Snippet: The mCherry/GFP nanobody (Nb) sequence was synthesized from Genescript (Nanjing) and cloned into the lentiCRISPRv2 vector. mCherry or green fluorescent protein (GFP) was amplified by PCR using primers for flanking and cloned into the psin-puromycin vector (Addgene) between MluI and SpeI restriction sites; CD63 was amplified by PCR, and the amplicons were cloned into psin-mCherry/GFP vector.

Techniques: Expressing, Membrane, Quantitative RT-PCR, Derivative Assay, Western Blot, RNA Immunoprecipitation, Immunoprecipitation, Control